The goal of this project is to define the molecular mechanisms involved in the replication of HIV and related retroviruses and in particular, to understand factors which influence early steps in virus infection. These studies are also directed toward development of new strategies for AIDS therapy. Our research is currently focused on two broad areas of interest: reverse transcription and virus assembly. During reverse transcription, there are two strand transfer events that are required for completion of plus- and minus-strand DNAs. Previously we demonstrated that the HIV-1 nucleocapsid protein (NC) is critical for efficient and specific HIV-1 minus-strand transfer; we now report that NC also plays an important role in the tRNA primer removal and annealing reactions in HIV-1 plus-strand transfer. Mutational analysis of NC function during both strand transfer events is currently underway, using NC mutants with changes in either one or both zinc fingers. Our earlier observation that actinomycin D interferes with NC activity in endogenous and reconstituted HIV-1 minus- strand transfer systems is now being pursued in cell culture studies, to determine whether treatment with actinomycin D alone or with other HIV inhibitors can reduce virus infectivity. The results will be used to evaluate the feasibility of clinical trials with AIDS patients. In work on virus assembly initiated during this past year, we are focusing on the role of the capsid protein (CA) in early post- entry events. Single- and double-mutations in conserved residues in the N-terminal portion of CA are being constructed. Thus far, several alanine-substitution mutants have been shown to retain the ability to produce virus particles; however, the virions are non-infectious. Further analysis of these mutants is in progress. - HIV, virus assembly, reverse transcription, nucleocapsid protein, actinomycinD, strand transfer